The YAK family of serine/threonine protein kinases represent a novel family of dual specificity protein kinases with unique structural, enzymatic, and probably functional features was identified (Becker and Joost (1999) Prog. Nucl. Acid Res. 62, 1–17). Four members of this subfamily have been identified by large scale screening of human cDNA libraries using yeast YAK1 sequence, and have been termed h (human)Yak1, 2, 3, and 4. See U.S. Pat. No. 5,972,606 (hYAK1), U.S. Pat. No. 6,001,623 (hYAK2), and U.S. Pat. No. 5,965,420 (hYAK3). In the yeast S. cerevisiae YAK1 functions as a negative regulator of cell growth (Garrett, S., Menold, M. M., and Broach, J. (1991) Mol. Cell. Biol. 11, 4045–4051). Deletion of the three PKA genes (tpk1, tpk2, and tpk3) in yeast causes cell cycle arrest at G1 while this growth defect is alleviated by removal of the YAK1 gene (Garrett, S., and Broach, J. (1989) Gene Dev. 3, 1336–1348). Recent data indicates that yYAK1 expression is controlled by two transcription factors MSN2/4 which are negatively regulated by PKA, thus yYAK1 acts downstream of PKA (Smith, A., Ward, M. P. and Garrett, S. (1998) EMBO J. 17, 3556–3564). While the means by which yYAK1 inhibits cell growth is still not known, overexpression of yYAK1 suppresses cell cycle arrest in late mitotic mutants activity (cdc15, cdc5, dbf2, and tem1) defective in anaphase-promoting complex (APC) (Jaspersen, S. L. Charles, J. F., Tinker-Kulberg, R. L., and Morgan, D. O. (1998) Mol. Biol. of the Cell. 9, 2803–2817). Recent work in Dictyostelium has uncovered a yYAK1 homolog which is required for the transition from growth to development giving support to the involvement of this family of kinases in cell growth (Souza, G. M., Lu, S. and Kuspa, A. (1998) Development 125, 2291–2302).
Human multi-tissue northern blot analysis indicated that hYAK1 is expressed as a ˜10 kb, 7.0 kb and 2.6 kb transcript. The multiple transcripts are not due to cross-hybridization with other YAK family members since the 3′UTR was used as a probe and the closest known homolog to hYAK1, hYAK3, shares only 62% identity with hYAK1 at the nucleotide level. In addition, alternatively spliced forms were identified within the 3′ UTR indicating that the multiple transcripts are due to alternative splicing within the untranslated regions. The most abundant transcripts were found in skeletal muscle and heart followed by pancreas, placenta, brain and lung. Multiple transcripts of the same apparent size were also seen in various osteoblastoid (HOS, MG63, Hob), stromal (TF274) and chondrocyte (C20A4) cell lines confirming that hYAK1 is expressed in these tissues. In situ hybridization studies were done using 35S-labeled riboprobes on cryosections of human bone and giant cell tumor. Autoradiographic development times were extended (3 weeks) to compensate for the generally low level of mRNA expression of hYAK1 kinase observed in the initial studies. In human fetal bone and osteophyte, various osteoblast populations were strongly (3+) positive for the expression of hYAK1 kinase mRNA. Many other cell types including bone marrow and chondrocytes had varying levels of expression (1–2+). In giant cell tumor, the diverse population of cell types including stromal, osteoblast precursors and osteoclasts were all positive (2+) for hYAK1 kinase expression.
Several lines of evidence from our research findings strongly suggest that hYAK1, like YAK1 in yeast functions as a negative regulator of cell cycle progression. Overexpression of wild type hYAK1 in cells causes a delay in exit from G2/M phase. Conversely, hYAK1 kinase inhibitors selectively cause an accumulation of S phase cells. This in turn causes changes in the expression of bone specific markers and products from chondrocytes. Specifically, YAK1 inhibitors are expected to increase bone formation and/or to be chrondroprotective.
Northern analysis was carried out to determine the distribution of hYAK3 mRNA in human tissues. Membranes containing mRNA from multiple human tissues (Clontech #7760-1, #7759-1, and #7768-1) were hybridized to an hYAK3 probe and washed under high stringency conditions as directed. Hybridized mRNA was visualized by exposing the membranes to X-ray film. One major transcript at ˜2.5 kb was present in testis, and transcripts of 2.5, 8 and 10 kb were present in bone and fetal liver. The transcripts were not visible in any other tissues; however, dot blot analysis using a Human Master blot (Clontech #7770-1) indicated that hYAK3 is expressed in other tissues including skeletal muscle.
Investigations with primary cells and hematopoietic cell lines from both human and mouse indicate that cells of the erythroid lineage may predominantly account for the elevated hYAK3 expression. These data suggest that hYAK3 may have lineage-specific function. In cell lines, hYAK3 is present at higher levels in cells with an erythroid phenotype than other hematopoietic lineages, including myeloid, monocytic and lymphoid cell lines. This profile is completely distinct from hYAK1 which has been observed only at low constitutive levels in hematopoietic cells and tissues. EPO-treatment of human bone marrow in vitro leads to induction and sustained expression of hYAK3 message and hYAK3 protein. Splenocytes from mice made anemic by phenylhydrazine treatment become enriched in erythroid progenitors and exhibit increased expression of hYAK3. Increases in both message and protein accompany induction of erythroid differentiation in UT7-EPO cells.
In yeast, yYAK is a negative regulator of growth via the cell cycle. Consequently, we would anticipate that hYAK3 participates in cell cycle control, and/or commitment to differentiation. We predict that an antagonist of hYAK3 would have a positive effect on cell growth. Our data indicates that it also may be involved in terminal differentiation and growth arrest in hematopoietic cells, especially in the erythroid lineage. Therefore compounds which antagonize YAK3 function or activity may be therapeutically useful in treating conditions of hematopoietic cellular deficiency, such as anemias, including anemias due to renal insufficiency or to chronic disease, such as autoimmunity or cancer, neutropenia, cytopenia, drug-induced anemias, polycythemia, cancer and myelosuppression.
It now has been discovered that a certain novel quinoline inhibitors of hYAK1 and/or hYAK3 kinases are useful for treating diseases of the erythroid and hematopoietic systems, including anemias due to renal insufficiency or to chronic disease, such as autoimmunity or cancer and drug-induced anemias, polycythemia, myelodysplastic syndrome, aplastic anemia and myelosuppression; cytopenia; neurodegeneration; and are also useful for controlling male fertility, especially for the purpose of achieving contraception.